JP2002521341A - Cow dung extract having anti-cancer and anti-inflammatory effects and method for producing the same - Google Patents

Abstract

Disclosed are bovine excretion extracts having anticancer and anti-inflammatory activity prepared by a process comprising the step of extracting bovine excretion with a polar solvent selected from the group consisting of lower alcohol, lower ketone, acetonitrile, water and mixtures thereof, a process for preparing the same, and the use thereof as an anticancer agent or adjuvant, or an anti-inflammatory agent.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a cow dung extract and a method for producing the same. More specifically, the present invention has an anticancer effect by inhibiting cell proliferation and antioxidation and an anti-inflammatory activity by inhibiting platelet aggregation, so that cow dung extract which can be usefully used as an anticancer agent, an anticancer adjuvant or an antiinflammatory agent The present invention relates to a product and a method for producing the same.

[0002] Cancer is a general term for malignant tumors in which a portion of cells begin to abnormally proliferate, and infiltrate surrounding normal tissues, continue to proliferate, and destroy the normal function of cells. Means Overgrowth results from mutations in gene expression that cause cells to escape all regulatory mechanisms.

[0003] Anticancer drugs that have been developed to date for treating cancer are chemical anticancer drugs that attack by targeting the difference in the proliferative power of cancer cells and normal cells, and the immune system that restores the body's immunity and builds cancer cells. They are broadly classified into anticancer agents (biological response modifiers).

[0004] Chemical anticancer agents are a kind of cytotoxic substance, and are effective in that cancer cells have a higher proliferation activity than normal cells. In other words, if a chemical anticancer drug that inhibits cell growth is administered to the human body, cancer cells having a strong growth activity will be more lethal than normal cells.

[0005] Chemical anticancer drugs developed to date fall into five categories: alkylating agents, antimetabolites, anticancer antibiotics, plant-derived anticancer drugs, and the like. An alkylating agent is an agent that exerts an anticancer effect by alkylating a hydrogen atom of a nucleic acid or protein. Antimetabolites are antagonists of furin or pyrimidine that inhibit DNA synthesis and exhibit anticancer effects. Anticancer antibiotics and plant-derived anticancer agents are anticancer agents derived from microorganisms and plants, respectively. Examples of other anticancer agents include L-asparaginase and cisflatin (a platinum-based compound).

[0006] As described above, chemical anticancer agents derived from microorganisms and plants are known as chemical anticancer agents having a cytostatic effect, but anticancer agents derived from animals have not been known so far.

[0007] Conventionally, animal excrement has been used by private companies for the treatment of various diseases. That is, human dung has traditionally been used for treatment purposes such as wounds or blood clots, and cow dung has been used for detoxification or burn treatment purposes. It is known in the animal world that injured animals eat cow dung. In spite of the fact that animal excrement has been widely used as a medicinal substance in this way, it is a fact that modern scientific studies have not so far elucidated any detailed research results. Furthermore, there is no known fact that animal excrement can be used as an anti-cancer or anti-cancer agent.

[0008] Accordingly, the present inventors have conducted scientific studies on the biological activity of cow dung in view of the above facts. As a result, the cow dung extract produced by the method according to the present invention has strong cell growth inhibition and antioxidant properties. The present inventors have found a new fact that they have an anticancer effect by an action and an anti-inflammatory effect by a platelet aggregation inhibitory action, and have completed the present invention.

An object of the present invention is to provide a bovine dung that can be usefully used as an anticancer agent, an anticancer adjuvant or an antiinflammatory agent by having an anticancer effect by suppressing cell proliferation and antioxidation and an antiinflammatory effect by inhibiting platelet aggregation. To provide an extract.

[0010] Yet another object of the present invention is to provide a method for producing cow dung extract.

The present invention relates to a method for converting cow dung into a lower alcohol having 1 to 4 carbon atoms, a lower ketone represented by the formula R ₁ COR ₂ (R ₁ and R ₂ are each independently an alkyl having 1 to 3 carbon atoms). , Acetonitrile, water and a mixture thereof, and a bovine dung extract having an anti-cancer and anti-inflammatory activity, which is produced by a method comprising extracting with a polar solvent selected from the group consisting of: The cow dung extract is a water-saturated (wa
It is preferably produced by a method further comprising a step of re-extracting with a non-polar organic solvent selected from the group consisting of tert-saturated butanol, ethyl acetate and ethyl ether, wherein the cow dung is bovine or buffalo feces. preferable.

[0012] The present invention further provides an anticancer agent comprising the above bovine feces extract as an active ingredient. The anti-cancer agent contains the bovine excrement extract in a single dose of 100 to 500 mg, and is preferably administered orally three times a day.

The present invention further provides an anti-inflammatory agent comprising an effective amount of the above-mentioned bovine feces extract as an active ingredient. The anti-inflammatory agent preferably contains the bovine feces extract in a single dose of 100 to 500 mg and is orally administered about three times a day.

The present invention relates to a method for converting cow dung into a lower alcohol having 1 to 4 carbon atoms, represented by the general formula R₁COR₂(R
₁And R₂Is each independently an alkyl having 1 to 3 carbons)
Selected from the group consisting of lower ketones, acetonitrile, water and mixtures thereof
Production of bovine feces extract with anti-cancer and anti-inflammatory activity including extracting with polar solvent
Provide a way. In the above production method, the substance obtained from the above step is
Non-polar organic solvent selected from the group consisting of alcohol, ethyl acetate and ethyl ether
Preferably, the method further comprises the step of re-extracting with.

Hereinafter, the present invention will be described in detail as follows. In the present invention, "
The term "bovine excretion" means stool of a bovine. The bovine is a concept including all animals belonging to the bovine family in the animal classification system, and in the present invention, a cow or a buffalo is more preferable. .

The cow dung extract according to the present invention is produced by first collecting fresh cow dung and extracting it with a polar solvent such as lower alcohol, lower ketone, acetonitrile, water or a mixture thereof. The lower alcohol is an alcohol having 1 to 4 carbon atoms, and is preferably methanol or ethanol. The lower ketone is represented by the formula R ₁ COR ₂ , wherein R ₁ and R ₂ are each independently alkyl having 1 to 3 carbon atoms, preferably acetone or methyl ethyl ketone.

According to the present invention, a cow dung extract is produced only by the above-mentioned extraction step, or a substance obtained from the above-mentioned extraction step is suspended in water, and then the non-polar extract such as water-saturated butanol, ethyl acetate and ethyl ether is used. It is prepared by re-extracting with an organic solvent.

The cow dung extract according to the present invention may contain various components derived from feed components consumed by cattle. Such components are present in the feed in its original or altered form. Such changes are produced by the metabolism of components consumed by the cow through the gastrointestinal tract of the cow and by the action of various digestive juices, enzymes or intestinal bacteria. Furthermore, the cow dung extract of the present invention also contains various digestive juice components secreted from the digestive tract of cattle.

The bovine feces extract of the present invention has an anticancer effect by inhibiting cell proliferation and antioxidation and an antiinflammatory effect by inhibiting platelet aggregation, and is useful for use as an anticancer agent, an anticancer adjuvant or an antiinflammatory agent. Can be. That is, an anticancer agent, an anticancer adjuvant or an anti-inflammatory agent containing the above-mentioned cow dung extract as an active ingredient can be produced. The above-mentioned drug is preferably orally administered in the present invention. However, the above-mentioned bovine feces extract is blended with a pharmaceutically acceptable carrier, and tablets, hard or soft capsules, liquids or suspensions common in the pharmaceutical field are used. It can be formulated into a preparation for oral administration such as a suspending agent. Pharmaceutically acceptable carriers include binders, lubricants, disintegrants, excipients, solubilizers,
Stabilizers, suspending agents, pigments, fragrances and the like can be mentioned. The anticancer or anti-inflammatory agent of the present invention is preferably administered in a single dose of 100 to 500 mg about three times a day. In the above case, the desired anticancer or anti-inflammatory effect can be sufficiently obtained. It is.

Examples Hereinafter, preferred examples of the present invention will be described. However, those skilled in the art will recognize that the particular embodiments described are merely illustrative of the invention, as described in more detail in the claims below, and are not and should not be considered limiting. It will be easy to understand.

Example 1 Production of cow dung extract 10 kg of fresh cow dung was added to 10 l of methanol, and the mixture was heated under reflux at a temperature of 60 ° C. for 1 hour. The extract thus obtained was filtered and the filtrate was depressurized to remove the organic solvent. Subsequently, the extract was extracted with water-saturated butanol to obtain 2.5 g of a non-polar component fraction, which was filtered and dried to obtain a powder. The cow dung extract thus obtained was analyzed using silica gel thin-layer chromatography with a mobile phase of chloroform / methanol = 50/1, and the results are shown in FIG. 1a. In FIG. 1a, spots indicate stigmastanol (C ₂₉ H ₅₂ O), and spots indicate beta-sitosterol (C ₂₉ H ₅₀ O).

Example 2 Production of cow dung extract 10 kg of fresh ethanol was added to 10 kg of fresh cow dung, stirred for 2 hours, and left to stand. The extract thus obtained was filtered to remove scum and precipitate, and the filtrate obtained was concentrated under reduced pressure and dried to obtain 3.0 g of a low-molecular-weight fraction powder. The cow feces extract thus obtained was subjected to a mobile phase of chloroform / methanol = 50 /
The results were analyzed using silica gel thin-layer chromatography, and the results were shown in FIG.
It was shown to. In FIG. 1b, the spots indicate stigmastanol (C ₂₉ H ₅₂ O),
Spots indicate beta-sitosterol (C ₂₉ H ₅₀ O), respectively.

Test Example 1 Cytotoxicity Measurement Test The cytotoxicity of the bovine feces extract according to the present invention was evaluated as the degree of hemolysis of erythrocytes as described below. The extract obtained in Example 1 was added to erythrocytes separated from the rat, and cultured at 37 ° C. for 30, 60 and 120 minutes, respectively. After centrifugation, 400 µl of each culture was taken, a hemolysis detection reagent was added, and the absorbance was measured at 540 nm to determine the degree of hemolysis. The degree of hemolysis (%) is defined as follows. * Degree of hemolysis (%) = (absorbance of supernatant / absorbance of 100% hemolyzed red blood cells) x 100

In order to compare the degree of hemolysis of the cow dung extract of Example 1, menadione was used.
The degree of hemolysis of the red blood cells (standard control) treated with was measured. Menadione is a chemical known to induce hemolysis of red blood cells. Table 1 shows the measurement results of cytotoxicity (cytotoxicity) indicated by the degree of hemolysis described above.

[0025]

[Table 1]

As shown in Table 1, menadione significantly increased the cytotoxicity due to erythrocyte hemolysis depending on the culture time, but the extract obtained in Example 1 showed a high concentration of 50 μg / ml.
However, erythrocyte hemolysis did not occur and did not show a significant difference from the control group. This result indicates that the cow dung extract according to the present invention is not toxic to cells.

Test Example 2 Measurement of Anticancer Effect by Method of Measuring Thymidine Incorporation The anticancer effect of the bovine feces extract obtained in Example 1 and Example 2 was measured using the following method for measuring the amount of thymidine introduced. DMEM (Dulbecco's Modified Eag
le's Medium, Gibco) 13.8 g was dissolved in 1 liter of deionized water, and the pH was adjusted to 7.4 using sodium carbonate and hydrochloric acid solution.
1 × 10 ⁻⁷ M insulin and gentamicin (50 mg / liter) were added, and the mixture was sterilized with a Millipore filter to prepare a culture solution. The culture solution was inoculated with human hepatocellular carcinoma sk-Hep-1 provided by Seoul National Cancer Institute at a rate of 1 × 10 ⁶ cells per 25 cm ² of T-flask area at 37 ° C. to maintain 5% carbon dioxide. For 48 hours.

After transferring this culture to a 24-well culture vessel and subculturing it for 1 day,
1 to 200 μg / ml of the cow dung extract obtained in Example 1 and Example 2 respectively
Was added. The extract obtained in Example 1 and Example 2 was treated to obtain 12
Time was labeled with ³ H after a lapse ⁽³ H-Labeled) 1 [mu] Ci of thymidine
/ Ml. After 12 hours again, the medium was removed from each well, the cells were fixed using methanol, and PBS (phosphate buffer) was removed.
ed saline) and washed twice with 10% trichloroacetic acid to remove unreacted radioactive thymidine. After lysing the cells with 1 M sodium hydroxide and neutralizing with 1 M hydrochloric acid, the radioactivity introduced into the DNA was measured with a flash counter (Pharmacia 1024).

The results are shown in FIGS. 2a and 2b, respectively. 2a and 2b are graphs showing the results when the cow dung extract obtained in Example 1 and Example 2 were used, respectively. The horizontal axis indicates the concentration of the feed, and the vertical axis indicates the amount of thymidine introduced. Things.

As shown in the above figures, the bovine feces extracts obtained in Examples 1 and 2 were shown to significantly reduce the amount of radioactive thymidine introduced at a concentration of 1 μg / ml or more. It was shown to inhibit sk-Hep-1 cell growth. Therefore, the bovine feces extract according to the present invention significantly suppresses the growth of cancer cells, and thus can be usefully used as an anticancer agent or an anticancer adjuvant.

Test Example 3 Measurement of Anti-Inflammatory Effect by the Method for Measuring Platelet Aggregation Inhibition Effect The anti-inflammatory effect of the extract obtained in Example 1 was measured using the following method for measuring platelet aggregation suppression effect. Platelet rich plasma (PRP) was adjusted to 3 × 10 ⁸ / ml platelets obtained from rat blood. 489 µl of PRP was placed in a free container for measuring platelet aggregation, and precultured at 37 ° C for 1 minute. 2.5 μl of the solution obtained by dissolving the extract obtained in Example 1 in methanol was added thereto. After 3 minutes, 8 μl of a thrombin solution was added, and then the platelet aggregation inhibitory action was measured using a platelet aggregometer. The results are shown in Table 2.

[0032]

[Table 2]

As shown in Table 2, the extract obtained in Example 1 strongly inhibited platelet aggregation. Therefore, the extract of the present invention is considered to be extremely useful as an anti-inflammatory agent.

The cow dung extract according to the present invention has an anticancer effect by inhibiting cell proliferation and antioxidation and an anti-inflammatory effect by inhibiting platelet aggregation, and thus is useful as an anticancer agent, an anticancer adjuvant or an antiinflammatory agent. Can be.

[Brief description of the drawings]

FIG. 1a is a drawing showing the results of analysis of the cow dung extract obtained from Example 1 by silica gel thin layer chromatography. FIG. 1b is a drawing showing the results of analysis of the cow dung extract obtained from Example 2 by silica gel thin layer chromatography. FIG. 2a is a graph showing the measurement results of the anticancer effect of the cow dung extract obtained from Example 1. FIG. 2b is a graph showing the measurement results of the anticancer effect of the cow dung extract obtained from Example 2.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Park Manki South Korea Shinwon Apartment 310-1502 (72) Inventor Park Jeong-Il South Korea Seoul Gangnam-gu Il-Wodong Hansol Apartment 301-208 (72) Inventor Lee Seunggi South Korea Seoul Seochog Bampodong 34-13 Hanshin Hangan Apartment 5-803 (72) Inventor Jeong Jinho Seoul South Korea Jangwondong Hanshin Apartment 347-806 (72) Inventor Park Seongwon South Korea Seoul Nryangu-time click 1 Don 20 Shin Ne deli-time apartment 505-601 F-term (reference) 4C087 BB33 BB70 CA06 MA52 NA14 ZB11 ZB26

Claims (9)

[Claims] 1. A cow dung is converted to a lower alcohol having 1 to 4 carbon atoms by the formula R ₁ COR ₂ (
R ₁ and R ₂ are each independently an alkyl having 1 to 3 carbon atoms), and a step of extracting with a polar solvent selected from the group consisting of lower ketones, acetonitrile, water and mixtures thereof. A cow dung extract having anti-cancer and anti-inflammatory activity to be produced. 2. The bovine feces extract according to claim 1, further comprising a step of re-extracting the extract with a non-polar organic solvent selected from the group consisting of water-saturated butanol, ethyl acetate and ethyl ether. 3. The cow dung extract according to claim 1, wherein the cow dung is bovine or buffalo feces. 4. An anticancer agent comprising, as an active ingredient, any of the cow dung extracts according to claim 1. 5. The anticancer agent according to claim 4, wherein the bovine excrement extract is contained in a single dose of 100 to 500 mg, and is orally administered about three times a day. 6. An anti-inflammatory agent comprising as an active ingredient any of the bovine feces extracts according to claim 1. 7. The anti-inflammatory agent according to claim 6, wherein the bovine excrement extract is contained in a single dose of 100 to 500 mg, and is orally administered about three times a day. 8. The cow dung is converted to a lower alcohol having 1 to 4 carbon atoms, a formula R ₁ COR ₂ (
R ₁ and R ₂ are each independently an alkyl having 1 to 3 carbon atoms), and a step of extracting with a polar solvent selected from the group consisting of lower ketones, acetonitrile, water and mixtures thereof. And a method for producing a cow dung extract having anti-inflammatory activity. 9. The method of claim 8, further comprising the step of re-extracting the extract with a non-polar organic solvent selected from the group consisting of water-saturated butanol, ethyl acetate and ethyl ether.